Sains Malaysiana 52(5)(2023): 1485-1496

http://doi.org/10.17576/jsm-2023-5205-12

 

Proportion of CD44+ Subset of Tumour Cells in Single Cell Suspension Prepared from FFPET Sections Directly Correlates with Histological Subtyping of Head and Neck Squamous Cell Carcinoma

(Bahagian CD44+ Subset Sel Tumor dalam Suspensi Sel Tunggal Disediakan daripada Bahagian FFPET Berkorelasi Terus dengan Pengetipan Histologi Karsinoma Sel Skuamosa Kepala dan Leher)

 

MUHAMMAD KASHIF1,2,*, SADIA MINHAS3, SHAH JAHAN1, AFRA SAMAD4, FAHEEM SHAHZAD1, ROMEEZA TAHIR1, ABDUL HANAN NAGI5 & NADEEM AFZAL1

 

1Department of Immunology, University of Health Sciences, 54000, Khyaban e Jamia Punjab, Lahore, Pakistan

2Department of Oral Pathology, Bakhtawar Amin Medical and Dental College, Northern Bypass Road, 60000, Multan, Pakistan

3Department of Oral Pathology, Akhtar Saeed Medical and Dental College, Bahria Town, 54000, Lahore, Pakistan

4Department of Histopathology, Multan Medical and Dental College, 60000, Southern bypass road, Multan, Pakistan

5Chughtai Laboratory, Lahore, Pakistan

 

Diserahkan: 19 Disember 2022/Diterima: 17 April 2023

 

Abstract

CD44 expression in tumours imparts potential to progress, metastasize, recurrence, and resistance against antineoplastic therapy. In this study, we sought to describe the variation in the immuno-expression and numeration of MDR1+ and CD44+ potential cancer stem cells in different histological grades and subtypes of head and neck squamous cell carcinoma (HNSCC). Flow-cytometric analysis was performed on single cell suspension prepared from formalin fixed paraffin embedded tissue (FFPET) sections of HNSCC using anti-CD44 and anti-MDR1/ABCB-1 primary monoclonal antibodies. Immunohistochemical (IHC) staining was also carried out using both of these antibodies on HNSCC tissue sections mounted on super frosted glass slides. On immunohistochemical analysis, the mean IRS for CD44 and MDR1 were 8.6364 ±3.02114 and 1.5909 ±1.27674 respectively. When mean immune-expression scores of CD44 antibody and MDR1/ABCB-1 were compared with histological grades and subtypes of HNSCC, the relationship was found to be statistically insignificant. Interestingly, a strong statistical difference (p = 0.000)  was observed when the mean score of subset of dysplastic squamous epithelial cells with characteristics of cell stemness (CD326+CD44+) was compared among different histological subtypes of HNSCC using flowcytometric analysis. While no statistically significant association was observed when the mean score for subset of dysplastic cells with potential of drug resistance (CD44+MDR1+) was compared among different histological subtypes of HNSCC. Although potential cancer stem cell marker CD44 and the multidrug resistance maker ABCB-1/MDR1 co-expressed in HNSCC but the proportion of CD326+CD44+ subset of tumour cells (potential cancer stem cells/CSCs) significantly correlates with least aggressive to more aggressive tumour subtypes.

 

Keywords: CD44; CSCs; FFPET; flowcytometry; HNSCC; immunohistochemistry; multidrug resistance; OSCC

 

Abstract

Pengekspresan CD44 dalam tumor memberikannya potensi untuk berkembang, bermetastasis, berulang dan berintangan terhadap terapi antineoplastik. Dalam kajian ini, kami ingin menerangkan variasi dalam pengekspresan imun dan penbilangan sel stem kanser berpotensi MDR1+ dan CD44+ dalam gred histologi yang berbeza dan pengetipan karsinoma sel skuamosa kepala dan leher (HNSCC). Analisis aliran-sitometri dilakukan pada penggantungan sel tunggal yang disediakan daripada bahagian tisu terbenam parafin tetap formalin (FFPET) HNSCC menggunakan antibodi monoklonal utama anti-CD44 dan anti-MDR1/ABCB-1. Pewarnaan imunohistokimia (IHC) juga dilakukan menggunakan kedua-dua antibodi ini pada bahagian tisu HNSCC yang dipasang pada slaid kaca super beku. Pada analisis imunohistokimia, purata IRS untuk CD44 dan MDR1 masing-masing ialah 8.6364 ±3.02114 dan 1.5909 ±1.27674. Apabila min skor pengekspresan imun antibodi CD44 dan MDR1/ABCB-1 dibandingkan dengan gred histologi dan pengetipan HNSCC, hubungan itu didapati tidak signifikan secara statistik. Menariknya, perbezaan statistik yang kuat (p = 0.000) diperhatikan apabila skor min subset sel epitelium skuamosa displastik dengan ciri-ciri pensteman sel (CD326+CD44+) dibandingkan antara pengetipan histologi HNSCC yang berbeza menggunakan analisis sitometri aliran. Walaupun tiada perkaitan yang signifikan secara statistik, diperhatikan apabila skor min bagi subset sel displastik dengan potensi rintangan drug (CD44+MDR1+) dibandingkan antara pengetipan histologi HNSCC yang berbeza. Walaupun penanda sel stem kanser berpotensi CD44 dan pembuat rintangan pelbagai drug ABCB-1/MDR1 dinyatakan bersama dalam HNSCC tetapi perkadaran subset CD326+CD44+ sel tumor (sel stem berpotensi kanser/CSC) berkorelasi dengan ketara dengan tumor yang paling tidak agresif kepada pengetipan tumor yang lebih agresif.

 

Kata kunci: CD44; CSCs; FFPET; HNSCC; imunohistokimia; OSCC; rintangan pelbagai ubat; sitometri aliran

 

RUJUKAN

Adelstein, D., Gillison, M.L., Pfister, D.G., Spencer, S., Adkins, D., Brizel, D.M., Burtness, B., BusseP.M., CaudellJ.J., Cmelak, A.J., Colevas, A.D., Eisele, D.W., Fenton, M., Foote, R.L., Gilbert, J., Haddad, R.I., Hicks, W.L., Hitchcock, Y.J., Jimeno, A., Leizman, D., Lydiatt, W.M., Maghami, E., Mell, L.K., Mittal, B.B., Pinto, H.A., Ridge, J.A., Rocco, J., Rodriguez, C.P., Shah, J.P., Weber, R.S., Witek, M., Worden, F., Yom, S.S.,  Zhen, W., Burns, J.L. & Darlow, S.D. 2017. NCCN guidelines insights: Head and neck cancers, Version 2. Journal of the National Comprehensive Cancer Network 15(6): 761-770. https://doi.org/10.6004/jnccn.2017.0101

Akhter, M., Hossain, S., Rahman, Q.B. & Molla, M.R. 2011. A study on histological grading of oral squamous cell carcinoma and its co-relationship with regional metastasis. Journal of Oral and Maxillofacial Pathology 15(2): 168. https://doi.org/10.4103/0973-029x.84485

Arantes, L.M.R.B., De Carvalho, A.C., Melendez, M.E. & Carvalho, A.L. 2017. Serum, plasma and saliva biomarkers for head and neck cancer. Expert Review of Molecular Diagnostics 18(1): 85-112. https://doi.org/10.1080/14737159.2017.1404906

Becker, M.  & Levy, D. 2017. Mod